EP3990033B1 - Gefriergetrocknete zusammensetzung zur herstellung von kalibrierten gasgefüllten mikrovesikeln - Google Patents

Gefriergetrocknete zusammensetzung zur herstellung von kalibrierten gasgefüllten mikrovesikeln Download PDF

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EP3990033B1
EP3990033B1 EP20761497.5A EP20761497A EP3990033B1 EP 3990033 B1 EP3990033 B1 EP 3990033B1 EP 20761497 A EP20761497 A EP 20761497A EP 3990033 B1 EP3990033 B1 EP 3990033B1
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freeze
microvesicles
drying
gas
calibrated
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French (fr)
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EP3990033A1 (de
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Anne LASSUS
Samir Cherkaoui
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Bracco Suisse SA
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Bracco Suisse SA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

  • the present invention generally relates to the field of ultrasound contrast-agents (USCA).
  • USCA ultrasound contrast-agents
  • a freeze-dried composition according to claim 1 which may be reconstituted for preparing a suspension of gas-filled microvesicles with calibrated size, useful in diagnostic or therapeutic applications. It further relates to the method for the preparation of such freeze-dried composition according to claim 8.
  • Calibrated-size microvesicles are a new generation of gaseous microbubbles having a narrow calibrated and controlled size distribution (mean sizes between 3 and 8 ⁇ m) compared to commercially available polydisperse microbubble ultrasound contrast-agents (USCA). These calibrated-size microbubbles are designed to enhance imaging sensitivity and improve efficiency to deliver drug and gene to specific organs. Calibrated microvesicles can be produced using various techniques: decantation, mechanical filtration, centrifugation, bubble sorting and flow-focusing.
  • the flow-focusing technology allows the manufacturing of calibrated microvesicles (typically with a geometric standard deviation (GSD) values between 1.05 and 1.08) in a highly reproducible way at a reasonable production rate ( ⁇ 60 million bubbles per minute), with acceptable concentrations of suspended microvesicles (e.g. between 3 ⁇ 10 8 CMV/mL and 4 ⁇ 10 8 CMV/mL) for subsequent uses.
  • GSD geometric standard deviation
  • Ref.1 [ WO2018/041906 A1 - BRACCO SUISSE SA ] and Ref.2 [PCT application number PCT/EP2019/055325 ], both in the name of the Applicant, describe a method for preparing CMV, such as gas-filled microbubbles, in particular by using microfluidic technique.
  • Freeze-drying also known as lyophilization, is a complex and challenging process, widely used in the pharmaceutical industry that has the advantage of preserving pharmaceutical products under a dry form over several months. In fact, freeze-dried products display greater storage stability and can be easily shipped. Freeze-drying is also used in the field of gas filled microvesicles for preparing freeze-dried dosage forms which are then reconstituted with an aqueous solvent in the presence of a gas, to form a suspension of gas-filled microvesicles.
  • one of the most challenging issue in preparing a freeze-dried composition of calibrated microvesicles is related to the need of avoiding substantial modification of the characteristics of the initial suspension of calibrated microvesicles, such as concentration, monodispersity or geometric standard deviation (GSD) and/or final mean diameter.
  • concentration concentration, monodispersity or geometric standard deviation (GSD) and/or final mean diameter.
  • GSD geometric standard deviation
  • the present invention relates to a freeze-dried composition according to claim 1 which, upon reconstitution with a pharmaceutically acceptable solution in the presence of a biocompatible gas, provides a suspension of calibrated gas-filled microvesicles, wherein said freeze-drying protecting component is a mixture of at least two freeze-drying protecting components and wherein said reconstituted suspension of calibrated gas-filled microvesicles have a geometric standard deviation (GSD) lower than 1.2.
  • GSD geometric standard deviation
  • the mixture of freeze-drying protecting components comprises a polyethylene glycol (PEG) and a polyol or a saccharide.
  • said mixture comprises a polyethylene glycol (PEG) and sorbitol or PEG and sucrose.
  • PEG polyethylene glycol
  • Said reconstituted suspension of calibrated microvesicles is characterized by a GSD of at least 1.2 or lower, preferably of at least 1.15, down to e.g. 1.1.
  • said reconstituted suspension of calibrated microvesicles is characterized by a concentration of at least 2.0 ⁇ 10 8 CMV/mL, preferably 2.25 ⁇ 10 8 CMV/mL, more preferably 2.5 ⁇ 10 8 CMV/mL, up to 5.5 ⁇ 10 8 CMV/mL.
  • the invention relates to a method of preparing a freeze-dried composition for the preparation of a reconstituted suspension of calibrated gas-filled microvesicles having a geometric standard deviation (GSD) lower than 1.2 according to claim 8.
  • GSD geometric standard deviation
  • a further aspect of the invention relates to a freeze-dried composition for preparing a suspension of calibrated gas-filled microvesicles, said freeze-dried composition being obtainable by a process according to claim 11.
  • a further aspect of the invention relates to a process for the preparation of an injectable contrast agent comprising a suspension of gas-filled microvesicles, wherein said process comprises reconstituting a freeze-dried composition as defined above, with a pharmaceutically acceptable solution in the presence of a biocompatible gas.
  • gas-filled microvesicles generally refers to bubbles of gas bounded, at the gas/liquid interface, by a very thin envelope (film) involving a stabilizing amphiphilic material comprising a phospholipid, disposed at the gas to liquid interface.
  • Said calibrated gas-filled microvesicles are suitable as contrast agents in ultrasound imaging techniques, known as Contrast-Enhanced Ultrasound (CEUS) Imaging, or in therapeutic applications, e.g. in combination with ultrasound mediated drug delivery.
  • CEUS Contrast-Enhanced Ultrasound
  • These stabilized gas bubbles are generally referred to in the art with various terminologies, depending typically from the stabilizing material employed for their preparation; these terms include, for instance, “microspheres”, “microbubbles”, “microcapsules” or “microballoons”, globally referred to here as “gas-filled microvesicles” (or “microvesicles” in short).
  • calibrated when referred to gas-filled microvesicles specifically refers to microvesicles suspensions with highly calibrated microvesicles (CMV), having different sizes between 3 and 8 ⁇ m, characterized by a size distribution having a geometric standard deviation (GSD) of at least 1.2 or lower, preferably of at least 1.1, down to e.g. 1.05.
  • CMS geometric standard deviation
  • calibrated gas filled microvesicles are preferably produced by using a microfluidic flow-focusing technology, where a gas thread is focused between two liquid flows in a flow-focusing device and phospholipid-stabilized calibrated microvesicles form and are collected in the outlet channel.
  • calibrated microvesicles are manufactured in a highly reproducible way at a reasonable production rate ( ⁇ 60 million bubbles per minute) ( Figure 2 and Figure 3 ).
  • the calibrated microvesicles may be obtained with relatively narrow size distribution around any desired mean diameter, e.g. from 3 to 8 ⁇ m, preferably about 4 ⁇ m.
  • the size distribution of said calibrated microvesicles is characterized by a geometric standard deviation (GSD) value of at least 1.2 or lower, preferably of at least 1.1, down to e.g. 1.05.
  • GSD geometric standard deviation
  • the calibrated microvesicles concentration (particularly upon production with microfluidic flow-focusing) is typically comprised between 3 ⁇ 10 8 and 4 ⁇ 10 8 CMV/mL, preferably close to 4 ⁇ 10 8 CMV/mL, not lower than 3 ⁇ 10 8 CMV/mL.
  • the "geometric standard deviation" generally provides a suitable value for characterizing the breath of the size distribution in a population of particles (gas-filled microvesicles in the specific case). A population of particles with a broad range of sizes will thus have a larger GSD value than one in which the particles sizes are narrowly distributed around a mean value (i.e.
  • Figure 1 shows an example of a size distribution graph (by volume) of a population of gas filled microvesicles which can be obtained with a commercial measuring device (e.g. Coulter Counter Multisizer 3, equipped with the Multisizer 3 software), by determining the volume of gas for each of its channels, each channel corresponding to a predetermined diameter of the microvesicles (e.g. with increments of 0.1 microns).
  • a commercial measuring device e.g. Coulter Counter Multisizer 3, equipped with the Multisizer 3 software
  • GSD e ⁇ n i ln x i ⁇ ln x ⁇ 2 ⁇ n i
  • the Coulter Counter Multisizer 3 equipped with the Multisizer 3 software, is capable of calculating and providing such GSD value as defined above.
  • a GSD value of 1.2 indicates that about the 50% of CMV are calibrated between 2.5 and 5 ⁇ m, for a mean diameter of 4 ⁇ m; a GSD of 1.05-1.08 ( ⁇ 1.1) indicates that about the 90-95% of CMV have sizes comprised between 2.5 and 5 ⁇ m.
  • microvesicles concentration refers to the number of CMV in a volume unit, determined using a Coulter Counter apparatus, i.e. number of CMV/mL.
  • said calibrated microvesicles obtained through microfluidic flow-focusing can be stored at room temperature for few weeks without substantial impacts on their main characteristics, after said storage period the characteristics of said microvesicles (e.g. concentration and size distribution) may not be maintained and may progressively deteriorate.
  • the lyophilization process is a suitable approach to obtain a calibrated microvesicles dry form, with increased stability over time and with preserved initial characteristics.
  • CMV initial characteristics such as concentration, GSD and final diameter
  • suitable combinations of freeze-drying protecting components as compared to a freeze-drying process using the same freeze-drying protecting components as single additive.
  • the present invention provides a freeze-dried composition according to claim 1, which upon reconstitution with a suitable aqueous solution in the presence of biocompatible gas, provides a suspension of calibrated gas-filled microvesicles, wherein said microvesicles have a GSD value of at least 1.2 or lower, preferably of at least 1.15, down to e.g. 1.1.
  • freeze-drying and “lyophilization” are used interchangeably, as well the terms “freeze-dried” and “lyophilized”.
  • freeze-dried composition indicates any dry dosage form for long-term storage of gas-filled microvesicles formulations obtained through a lyophilization process.
  • Said freeze-dried composition can comprise one or more active ingredient and a mixture of at least two freeze-drying protecting components.
  • active ingredient indicates the microvesicle-stabilizing materials, e.g. the amphiphilic materials, which are comprised in the freeze-dried composition with the freeze-drying protecting components.
  • mixture of freeze-drying protecting components refers to a combination of at least two components suitable for freeze drying, comprising a polyethylene glycol and a polyol or a saccharide which is included in the microvesicles suspension before the lyophilization process thereof.
  • freeze-drying protecting components designates any compound added to protect the active ingredient during any phase of the freeze-drying process.
  • suitable freeze-drying protecting components are polyethylene glycols (PEG), polyols, saccharides, surfactants, buffers, amino acids, chelating complexes, and inorganic salts.
  • said mixture of freeze-drying protecting components is a combination of at least two different compounds suitable for freeze-drying, whereby said mixture comprises a combination of a polyethylene glycol with a polyol or a saccharide.
  • Polyethylene glycol has its standard chemical meaning.
  • the chemical formula of PEG is HOCH 2 (CH 2 OCH 2 ) m CH 2 OH where m represents the average number of oxyethylene groups.
  • the expression “molecular weight” indicates the average length of the PEG polymer chains, with a variability of ⁇ 10% on the indicated molecular weight.
  • the mixture of freeze-drying protecting components preferably comprises a PEG having a molecular weight comprised between 2000 and 10000 g/mol, preferably between 4000 and 8000 g/mol.
  • the freeze-drying protecting component is PEG having a molecular weight of 8000 g/mol ( ⁇ 10%). According to another embodiment, the freeze-drying protecting component is PEG having a molecular weight close to 4000 g/mol 10%).
  • the mixture of freeze-drying protecting components is added to the suspension of CMV as a solution having a concentration comprised between 120 mg/mL and 250 mg/mL, more preferred close to 200 mg/mL.
  • polymers in particular polyglycol, e.g. PEG
  • concentration of 150 mg/mL or higher e.g. 200 mg/mL
  • PEG polyglycol
  • the Applicant has found that it is preferred using a suitable mixture of PEG at a concentration lower than 150 mg/mL (e.g. 100 mg/mL) in combination with a second freeze-drying protecting component.
  • the freeze-dried composition comprises a mixture of freeze-drying protecting components which is the combination of a polyethylene glycol with a polyol.
  • polyol has its conventional chemical meaning; it indicates any organic compound with more than two hydroxyl functional groups, characterized by the general formula HOCH 2 (CHOH) n CH 2 OH, where n is an integer from 1 to 6, preferably from 2 to 4. Polyols differ in chain length, i.e. four-, five- or six-carbon chains. They have one hydroxyl group attached to each carbon. Polyols can be further differentiated by the relative orientation (stereochemistry) of these hydroxyl groups. Suitable polyols include erythritol, xylitol, sorbitol, lactitol and mannitol.
  • said polyol is preferably selected from the group of polyols having a carbon chain length from four to six carbons atoms.
  • said polyol is sorbitol or xylitol.
  • Sorbitol has its conventional meaning in the chemical field.
  • Sorbitol, or (2R,3R,4R,5S)-hexane-1,2,3,4,5,6-hexol is a polyhydric alcohol, consisting in a linear carbon chain, with 6 carbon atoms, each one is substituted with a hydroxyl functional group. It has a molecular weight of 182.172 g/mol. Sorbitol occurs naturally and is also produced synthetically from glucose. It is isomeric with mannitol.
  • xylitol has its conventional meaning in the chemical field.
  • the freeze-dried composition comprises a mixture of a polyethylene glycol with a saccharide.
  • saccharides also called carbohydrates, are molecular compounds made from just three elements: carbon, hydrogen and oxygen.
  • the simplest saccharides are called monosaccharides and they are the building units for bigger saccharides, such as disaccharides, trisaccharide and polysaccharides.
  • said saccharide is selected from the group of disaccharides, trisaccharides and polysaccharides, more preferably a disaccharide or trisaccharide.
  • Monosaccharides have the general molecular formula (CH 2 O) n , where n can be 3, 5 or 6. Monosaccharides can form cyclic structures by the reaction of the carbonyl group with an OH group. These cyclic molecules can in turn react with another alcohol. Suitable examples of monosaccharides include glucose, fructose and galactose.
  • Disaccharides (C 12 H 22 O 11 ) are sugars composed of two monosaccharide units that are joined by a glycosidic bond. This latter is a covalent bond formed from the reaction of the anomeric carbon of one cyclic monosaccharide with the OH group of a second monosaccharide. Disaccharides differ from one another in their monosaccharide constituents and in the specific type of glycosidic linkage connecting them. Examples of disaccharides include: maltose, lactose, and sucrose. Particularly preferred among the disaccharides is sucrose.
  • Trisaccharides are saccharides composed of three monosaccharides with two glycosidic bonds connecting them. Similar to the disaccharides, each glycosidic bond can be formed between any hydroxyl group on the component monosaccharides. Even if all three component sugars are the same (e.g., glucose), different bond combinations (regiochemistry) and stereochemistry (alpha- or beta-) result in trisaccharides that are diastereoisomers with different chemical and physical properties. Examples of trisaccharides are maltotriose, melezitose, maltotriulose and raffinose. Particularly preferred among the trisaccharides is raffinose.
  • Polysaccharides are polymeric saccharide molecules composed of long chains of monosaccharide units bound together by glycosidic linkages. They range in structure from linear to highly branched. Examples of polysaccharides are: starch, cellulose, dextran and chitin. Particularly preferred among the polysaccharides is dextran.
  • said saccharide is a disaccharide, more preferably sucrose.
  • sucrose is its standard meaning.
  • Sucrose is a disaccharide formed by glucose and fructose units linked by an acetal oxygen bridge from hemiacetal of glucose to the hemiketal of the fructose.
  • Sucrose has an empirical formula of C 12 H 22 O 11 and a molecular weight of 342.30 g/mol.
  • Initial CMV characteristics are particularly preserved when using a mixture of freeze-drying protecting components characterized in that said mixture of freeze-drying protecting component has a total concentration comprised between 120 mg/mL and 250 mg/mL, more preferably the total concentration of PEG and polyol or PEG and saccharide is 200 mg/mL.
  • the mixture of freeze-drying protecting components comprises a PEG and a polyol or a PEG and a saccharide in a ratio between 2:1 to 2:3, preferably between 3:2 to 4:5, more preferably 1:1.
  • microvesicle-stabilizing materials Materials suitable for forming the stabilizing layer of the gas-filled microvesicle are those known in the art. These are amphiphilic materials.
  • amphiphilic material includes compounds having a molecule with a hydrophilic polar head portion (e. g. a polar or ionic group), capable of interacting with an aqueous medium, and a hydrophobic organic tail portion (e. g. a hydrocarbon chain), capable of interacting with e. g. an organic solvent.
  • hydrophilic polar head portion e. g. a polar or ionic group
  • hydrophobic organic tail portion e. g. a hydrocarbon chain
  • Suitable amphiphilic materials comprise phospholipids; lysophospholipids; fatty acids, such as palmitic acid, stearic acid, arachidonic acid or oleic acid; lipids bearing polymers, such as chitin, hyaluronic acid, polyvinylpyrrolidone or polyethylene glycol (PEG), also referred as "pegylated lipids"; lipids bearing sulfonated mono- di-, oligo- or polysaccharides; cholesterol, cholesterol sulfate or cholesterol hemisuccinate; tocopherol hemisuccinate; lipids with ether or ester-linked fatty acids; polymerized lipids; diacetyl phosphate; dicetyl phosphate; ceramides; polyoxyethylene fatty acid esters (such as polyoxyethylene fatty acid stearates), polyoxyethylene fatty alcohols, polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan
  • the amphiphilic material is a phospholipid.
  • phospholipid is intended to encompass any amphiphilic phospholipidic compound, the molecules of which are capable of forming a stabilizing film of material (typically in the form of a mono-molecular layer) at the gas-water boundary interface in the final microbubbles suspension. Accordingly, these materials are also referred to in the art as" film-forming phospholipids.
  • Suitable phospholipids include esters of glycerol with one or preferably two (equal or different) residues of fatty acids and with phosphoric acid, wherein the phosphoric acid residue is in turn bound to a hydrophilic group, such as, for instance, choline (phosphatidylcholines - PC), serine (phosphatidylserines - PS), glycerol (phosphatidylglycerols - PG), ethanolamine (phosphatidylethanolamines - PE), inositol (phosphatidylinositol).
  • choline phosphatidylcholines - PC
  • serine phosphatidylserines - PS
  • glycerol phosphatidylglycerols - PG
  • ethanolamine phosphatidylethanolamines - PE
  • inositol phosphatidylinositol
  • Esters of phospholipids with only one residue of fatty acid are generally referred to in the art as the "lyso" forms of the phospholipid or "lysophospholipids".
  • Fatty acids residues present in the phospholipids are in general long chain aliphatic acids, typically containing from 12 to 24 carbon atoms, preferably from 14 to 22; the aliphatic chain may contain one or more unsaturations or is preferably completely saturated.
  • suitable fatty acids included in the phospholipids are, for instance, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid.
  • saturated fatty acids such as myristic acid, palmitic acid, stearic acid and arachidic acid are employed.
  • phospholipids are phosphatidic acids, i.e. the diesters of glycerol-phosphoric acid with fatty acids; sphingolipids such as sphingomyelins, i.e. those phosphatidylcholine analogs where the residue of glycerol diester with fatty acids is replaced by a ceramide chain; cardiolipins, i.e. the esters of 1,3-diphosphatidylglycerol with a fatty acid; glycolipids such as gangliosides GM1 (or GM2) or cerebrosides; glucolipids; sulfatides and glycosphingolipids.
  • phosphatidic acids i.e. the diesters of glycerol-phosphoric acid with fatty acids
  • sphingolipids such as sphingomyelins, i.e. those phosphatidylcholine analogs where the residue of glycerol diester with fatty acids
  • phospholipids include either naturally occurring, semisynthetic or synthetically prepared products that can be employed either singularly or as mixtures.
  • phospholipids examples include natural lecithins (phosphatidylcholine (PC) derivatives) such as, typically, soya bean or egg yolk lecithins.
  • PC phosphatidylcholine
  • Examples of semisynthetic phospholipids are the partially or fully hydrogenated derivatives of the naturally occurring lecithins.
  • Preferred phospholipids are fatty acids diesters of phosphatidylcholine, ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol or of sphingomyelin.
  • phospholipids are, for instance, dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), diarachidoyl-phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine (DOPC), 1,2 Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC), dipentadecanoyl-phosphatidylcholine (DPDPC), 1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), 1-stearoyl-2-palmito
  • Suitable phospholipids further include phospholipids modified by linking a hydrophilic polymer, such as polyethyleneglycol (PEG) or polypropyleneglycol (PPG), thereto.
  • PEG polyethyleneglycol
  • PPG polypropyleneglycol
  • Preferred polymer-modified phospholipids include "pegylated phospholipids", i.e. phospholipids bound to a PEG polymer.
  • pegylated phospholipids are pegylated phosphatidylethanolamines ("PE-PEGs" in brief) i.e. phosphatidylethanolamines where the hydrophilic ethanolamine moiety is linked to a PEG molecule of variable molecular weight (e.g.
  • DPPE-PEG2000 refers to DPPE having attached thereto a PEG polymer having a mean average molecular weight of about 2000.
  • Particularly preferred phospholipids are DAPC, DSPC, DPPC, DMPA, DPPA, DSPA, DMPG, DPPG, DSPG, DMPS, DPPS, DSPS and Ethyl-DSPC. Most preferred are DPPG, DPPS and DSPC.
  • Mixtures of phospholipids can also be used, such as, for instance, mixtures of DPPE and/or DSPE (including pegylated derivatives), DPPC, DSPC and/or DAPC with DSPS, DPPS, DSPA, DPPA, DSPG, DPPG, Ethyl-DSPC and/or Ethyl-DPPC.
  • a mixture of phospholipids may include phosphatidylcholine derivatives, phosphatidic acid derivatives and pegylated phosphatidylethanolamine, e.g. DSPC/DPPA/DPPE-PEG, DPPC/DPPA/DPPE-PEG, DSPC/DPPA/DSPE-PEG, DPPC/DPPA/DSPE-PEG, DAPC/DPPA/DPPE-PEG, DAPC/DPPA/DSPE-PEG, DSPC/DSPA/DPPE-PEG, DPPC/DSPA/DSPE-PEG, DSPC/DSPG/DPPE-PEG, DPPC/DSPG/DSPE-PEG.
  • DSPC/DPPA/DPPE-PEG DPPC/DPPA/DPPE-PEG
  • DSPC/DPPA/DPPE-PEG DPPC/DPPA/DSPE-PEG
  • DPPC/DPPA/DSPE-PEG DPPC/DPPA/DSPE-
  • the phospholipid can conveniently be used in admixtures with any of the above listed amphiphilic compounds.
  • lipids such as cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate or ascorbyl palmitate, fatty acids such as myristic acid, palmitic acid, stearic acid, arachidic acid and derivatives thereof or butylated hydroxytoluene and/or other non-phospholipid compounds can optionally be added to one or more of the foregoing phospholipids, e.g in proportions preferably ranging from zero to 50% by weight, more preferably up to 25%.
  • mixtures of amphiphilic materials comprising phospholipids and fatty acids can advantageously be used, including DSPC/DPPG/palmitic acid, DSPC/DPPE-PEG/palmitic acid, DPPC/DPPE-PEG/palmitic acid, DSPC/DSPE-PEG/palmitic acid, DPPC/DSPE-PEG/palmitic acid, DSPC/DPPE-PEG/stearic acid, DPPC/DPPE-PEG/stearic acid, DSPC/DSPE-PEG/stearic acid or DPPC/DSPE-PEG/stearic acid.
  • microvesicles prepared according to the invention may optionally comprise a targeting ligand.
  • targeting ligand includes within its meaning any compound, moiety or residue having, or being capable to promote, a targeting activity (e.g. including a selective binding) of the microvesicles of a composition of the invention towards any biological or pathological site within a living body.
  • Targets with which targeting ligand may be associated include tissues such as, for instance, myocardial tissue (including myocardial cells and cardiomyocytes), membranous tissues (including endothelium and epithelium), laminae, connective tissue (including interstitial tissue) or tumors; blood clots; and receptors such as, for instance, cell-surface receptors for peptide hormones, neurotransmitters, antigens, complement fragments, and immunoglobulins and cytoplasmic receptors for steroid hormones.
  • tissues such as, for instance, myocardial tissue (including myocardial cells and cardiomyocytes), membranous tissues (including endothelium and epithelium), laminae, connective tissue (including interstitial tissue) or tumors; blood clots; and receptors such as, for instance, cell-surface receptors for peptide hormones, neurotransmitters, antigens, complement fragments, and immunoglobulins and cytoplasmic receptors for ste
  • the targeting ligand may be synthetic, semi-synthetic, or naturally occurring.
  • Materials or substances which may serve as targeting ligands include, for example, but are not limited to proteins, including antibodies, antibody fragments, receptor molecules, receptor binding molecules, glycoproteins and lectins; peptides, including oligopeptides and polypeptides; peptidomimetics; saccharides, including mono and polysaccharides; vitamins; steroids, steroid analogs, hormones, cofactors, bioactive agents and genetic material, including nucleosides, nucleotides and polynucleotides.
  • the targeting ligand may be an amphiphilic compound per se (which is admixed with the other components of the microvesicle) or a compound bound to an amphiphilic molecule (e.g. a phospholipid) employed for the formation of the microvesicles.
  • an amphiphilic compound per se which is admixed with the other components of the microvesicle
  • a compound bound to an amphiphilic molecule e.g. a phospholipid
  • Suitable gases comprise biocompatible fluorinated gases, preferably perfluorinated gases.
  • Fluorinated gases include materials which contain at least one fluorine atom such as, for instance, fluorinated hydrocarbons (organic compounds containing one or more carbon atoms and fluorine); sulfur hexafluoride; fluorinated, preferably perfluorinated, ketones such as perfluoroacetone; and fluorinated, preferably perfluorinated, ethers such as perfluorodiethyl ether.
  • Preferred compounds are perfluorinated gases, such as SF 6 or perfluorocarbons (perfluorinated hydrocarbons), i.e. hydrocarbons where all the hydrogen atoms are replaced by fluorine atoms, which are known to form particularly stable gas-filled microvesicles suspensions.
  • perfluorocarbon includes saturated, unsaturated, and cyclic perfluorocarbons.
  • biocompatible, physiologically acceptable perfluorocarbons are: perfluoroalkanes, such as perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes (e.g. perfluoro-n-butane, optionally in admixture with other isomers such as perfluoro-isobutane), perfluoropentanes, perfluorohexanes or perfluoroheptanes; perfluoroalkenes, such as perfluoropropene, perfluorobutenes (e.g.
  • perfluorobut-2ene or perfluorobutadiene
  • perfluoroalkynes e.g. perfluorobut-2-yne
  • perfluorocycloalkanes e.g. perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentanes, perfluorocyclohexane, perfluoromethylcyclohexane and perfluorocycloheptane).
  • Preferred saturated perfluorocarbons include, for example, CF 4 , C 2 F 6 , C 3 F 8 , C 4 F 8 , C 4 F 10 , C S F 12 and C 6 F 14 .
  • Particularly preferred gases are those which are in gaseous form at room temperature, including SF 6 , C 3 F 8 , and C 4 F 10 .
  • An embodiment thus relates to a method of preparing a freeze-dried composition for long term storage of calibrated gas-filled microvesicles, comprising the steps of:
  • the method of preparation of step a) is the microfluidic flow-focusing technique, as illustrated in Ref.1 [ WO2018/041906 A1 - BRACCO SUISSE SA ] and Ref.2 [PCT application number PCT/EP2019/055325 ].
  • FIG. 2 shows a schematic representation of the core portion 200 of a flow-focusing device ("microfluidic chip") useful in the process of the invention.
  • the chip comprises a first feed channel 201 for feeding the gaseous flow 201' and two additional orthogonal feed channels 202a and 202b for supplying the liquid flow comprising the amphiphilic material.
  • the gas flow and the two liquid flows are directed towards the contact zone 203 and then through the calibrated orifice 204, shown as a dotted line in figure 1 .
  • the calibrated orifice is connected to a calibrated channel 204' having preferably the same cross-section as the orifice, which is in turn connected to an initial portion 205 of the outlet channel 206.
  • the calibrated orifice 204 may be a nozzle directly connected to the initial portion 205 of outlet channel 206 i.e. without the calibrated channel in-between.
  • the microvesicles 203' are formed in the calibrated orifice and directed, through calibrated channel 204', to the initial portion 205 of the outlet channel 206.
  • the hydraulic diameter of the outlet channel is generally larger than the hydraulic diameter of the calibrated orifice and typically increases from the initial diameter of the calibrated orifice to the final diameter of the outlet channel 206, corresponding substantially to the hydraulic diameter of a collecting tube (not shown), connecting the flow-focusing device to a container, e.g. a sealed vial for collecting the suspension of microvesicles.
  • the temperature of the microvesicles is controlled, as described in Ref.1 [ WO2018/041906 A1 - BRACCO SUISSE SA ] and Ref.2 [PCT application number PCT/EP2019/055325 ].
  • the aqueous liquid flow for preparing the calibrated gas-filled microvesicles according to the method of the invention comprises an amphiphilic material (as above defined) at a concentration of e.g. from 5.0 to 20 mg/mL, preferably from 7.5 to 15 mg/mL, dispersed in an aqueous carrier.
  • Suitable aqueous carriers which are preferably physiologically acceptable, comprise water (preferably sterile water), aqueous solutions such as saline (which may advantageously be balanced so that the final product for injection is not hypotonic), or solutions of one or more tonicity adjusting substances.
  • Tonicity adjusting substances comprise salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials (e.g.
  • chitosan derivatives such as carboxymethyl chitosan, trimethyl chitosan or gelifying compounds, such as carboxymethylcellulose, hydroxyethyl starch or dextran.
  • an additional oil phase may be added for incorporating therapeutic hydrophobic substances into the microvesicles.
  • two additional conduits may be provided in the device for supplying the desired oil phase, as described for instance by by Ref.1 [ WO2018/041906 A1 - BRACCO SUISSE SA ] and Ref.2 [PCT application number PCT/EP2019/055325 ].
  • the formed gas-filled microvesicles will thus have a film of oil disposed at the interface between gas and the stabilizing layer of amphiphilic material, which can be loaded with a desired therapeutic agent.
  • Suitable oils may include any biocompatible oil which is liquid at room temperature including, for instance, mono-, di- or tri-esters of glycerol with saturated or unsaturated (C 2 -C 18 ) alkyl chains (including homo- or hetero-allkylesters), such as glycerol monobutyrin, glycerol monolinoleate, 1,2-dihexanoyl glycerol, 1,2 dioctanoyl glycerol, 1,2-dioleyl-sn-glycerol, triacetin, tributyrin, tricaproin, tricaprylin, tricaprin, and mixtures thereof; or natural oils such as soya oil, olive oil, safflower seed oil, sunflower seed oil, peanut oil and mixtures thereof.
  • biocompatible oil which is liquid at room temperature including, for instance, mono-, di- or tri-esters of glycerol with saturated or unsaturated (C 2 -C 18 ) alkyl
  • the freshly formed microvesicles comprise a gas selected among those previously indicated.
  • the gas is a mixture of a gas highly soluble in water (“HS gas”) and of a gas with low solubility in water (“LS gas”), as anticipated in Ref. 2 [PCT application number PCT/EP2019/055325 ].
  • the major amount of the highly soluble gas rapidly dissolves in water while the poorly soluble one remains entrapped into the densely packed layer of amphiphilic compounds, typically with some residual amount of HS solubility gas dispersed therein.
  • HS gases examples include nitrogen, air, and carbon dioxide, this latter being particularly preferred because of its higher solubility in water.
  • Suitable LS gases are fluorinated gases, preferably perfluorinated gases. Fluorinated gases those previously described in the present description.
  • gas-filled microvesicles comprising CO 2 /C 4 F 10 in a volume ratio of from 80/20 to 90/10, e.g. 85/15, can be prepared with a gas-mixing device similar to the one schematically illustrated in Figure 3 .
  • Figure 3 shows an example of a microfluidic flow-focusing device used for the production of calibrated microvesicles.
  • a gas flow 302 (comprising e.g. a mixture of C 4 F 10 and COz) and a liquid flow 301 (comprising an amphiphilic material, e.g. a phospholipid, fatty acid or mixtures thereof), are supplied to microfluidic chip 303 to produce microvesicles through orifice 304.
  • the microvesicles suspension is collected in a vial 305, which is preferably prefilled with a gas (e.g. C 4 F 10 ) at ambient pressure.
  • a venting device (e.g.
  • a needle 306 is preferably used to equalize the overpressure generated by the liquid filling of the vial.
  • the venting device is preferably removed and the container is preferably sealed to avoid further gaseous exchange with the external atmosphere.
  • the calibrated microvesicles obtained through the microfluidic flow-focusing method are treated using suitable washing techniques, in order to remove not-assembled amphiphilic material and possible residue compounds.
  • washing indicates any operation carried out on the freshly prepared microvesicles suspension, finalized to remove (or substantially reduce the amount of) not-assembled amphiphilic material and residue compounds.
  • suitable washing techniques comprise centrifugation, filtration, bubble sorting and decantation.
  • not-assembled amphiphilic material indicates amphiphilic molecules that, at the end of the preparation process, are present in the calibrated microvesicles suspension, but are not forming the stabilizing layer of the gas-filled microvesicles.
  • residues compounds indicate any possible additive substance that is added to the amphiphilic material solution during the microvesicles preparation, such as tonicity adjusters as described before.
  • a mixture of freeze-drying protecting components is added to the calibrated microvesicles suspension.
  • the mixture of freeze-drying protecting components can be added to the liquid flow comprising the amphiphilic compounds, described above, during the preparation of the microvesicles by microfluidic technique.
  • Initial CMV characteristics are particularly preserved when using a mixture of freeze-drying protecting components characterized in that said mixture of freeze-drying protecting component has a total concentration comprised between 120 mg/ml and 250 mg/mL, more preferably the total concentration of PEG and polyol or PEG and saccharide is 200 mg/mL.
  • the CMV suspension before the freeze-drying process, comprises a mixture of freeze-drying protecting components at a concentration between 10 and 25%, preferably between 14 and 24% and still more preferred between 18-22% (w/v%).
  • the mixture of freeze-drying protecting components represents the larger amount of the final freeze-dried preparation, wherein it is typically at least 90%, preferably between 94% and 99.7%, more preferably 99.5%, up to 99.9% (w/w).
  • Said mixtures of freeze-drying protecting components are those described above in the present description. Said mixture has shown advantageous results when used in the freeze-drying process of the calibrated microvesicles suspensions, to prepare a freeze-dried composition which can then be reconstituted to obtain suspensions of calibrated microvesicles having acceptable characteristics in terms of concentration and size distribution, as compared to those of the initial suspension (before freeze-drying).
  • GSD values comprised between 1.16 and 1.18 can be obtained when using a mixture of freeze-drying protecting components, in comparison with the use of a single additive which gives calibrated microvesicles characterized by a higher GSD value.
  • the use of a mixture of freeze-drying protecting components is able to significantly improve the yield of calibrated microvesicles after freeze-drying with an increase of 38%, when compared with the use of a single freeze-drying protecting component.
  • freeze-drying has its standard meaning in the pharmaceutical technology field. Freeze-drying process consists of drying a pre-frozen liquid product under low pressure or vacuum and at low temperature. The main objective is to remove liquid from the product in order to provide a freeze-dried product suitable for long term storage.
  • freeze-drying parameters may be selected to further optimize the characteristics of the reconstituted suspension of microvesicles (e.g. yield, size, GSD).
  • the freezing temperature of said method for long term storage of calibrated gas-filled microvesicles ranges between -30 °C and - 70 °C, preferably between -30 °C and -60 °C, still more preferably -40°C.
  • the pressure of the freeze-drying is preferably 0.5 mbar or lower, preferably 0.2 or lower, e.g. close to 0.1 mbar.
  • a further aspect of the invention relates to a freeze-dried composition for preparing a suspension of calibrated gas-filled microvesicles, said freeze-dried composition being obtainable by a process according to claim 11.
  • a further aspect of the invention relates to a process for the preparation of an injectable contrast agent comprising a suspension of gas-filled microvesicles, wherein said process comprises reconstituting a freeze-dried composition, obtained as described above, comprising an amphiphilic material and a mixture of freeze-drying protecting components, with a pharmaceutically acceptable solution in the presence of biocompatible gas.
  • the freeze-dried composition can then be reconstituted with a suitable pharmaceutically acceptable (aqueous) solution in the presence of biocompatible gas, thus providing a suspension of calibrated gas-filled microvesicles, wherein said microvesicles have a GSD of at least 1.2 or lower, preferably of at least 1.15, down to e.g. 1.1.
  • aqueous solutions are water, typically sterile, pyrogen free water (to prevent as much as possible contamination in the final reconstituted product), aqueous solutions such as saline (which may advantageously be balanced so that the final product for injection is not hypotonic), or aqueous solutions of one or more tonicity adjusting substances such as salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials.
  • the freeze-dried composition is typically reconstituted with a volume of aqueous solution similar to the volume of suspension which underwent the freeze-drying process. Accordingly, the concentration of the freeze-drying protecting components in the reconstituted suspension is substantially the same as the one in the initial suspension. For this reason, an excessive amount of polymer (typically polyglycol, e.g. PEG), e.g. higher than 150 mg/mL (e.g. 200 mg/mL) shall preferably be avoided, in order to avoid an excessive viscosity of the suspension to be administered.
  • polyglycol e.g. PEG
  • 150 mg/mL e.g. 200 mg/mL
  • the reconstituted suspensions of calibrated microvesicles were found to have substantially maintained the initial characteristics of calibrated microvesicles, as characterized before the lyophilization process, resulting suitable for subsequent pharmaceutical uses.
  • Said reconstituted suspension of calibrated microvesicles is characterized by calibrated microvesicles having a GSD of at least 1.2 or lower, preferably of at least 1.15, down to e.g. 1.1.
  • said reconstituted suspension of calibrated microvesicles is characterized by a concentration of at least 2.0 ⁇ 10 8 CMV/mL, preferably 2.25 ⁇ 10 8 CMV/mL, more preferably 2.5 ⁇ 10 8 CMV/mL, up to 5.50 ⁇ 10 8 CMV/mL.
  • microvesicles concentration refers to the number of microvesicles in a volume unit, determined using a Coulter Counter apparatus, i.e. number of MB/mL.
  • the concentration of calibrated microvesicles (%), measured after the reconstitution of the freeze-dried composition of the invention with suitable aqueous solution allows to determine the microbubble yield after said reconstitution, in comparison to the microvesicles concentration measured before the freeze-drying process.
  • the calibrated microvesicles yield after the reconstitution of the freeze-dried composition of the invention is at least 50%, preferably at least 55%, more preferably at least 60% even more preferably at least 65%, up to e.g. 85%, preferably 90%, more preferably 95%, even more preferably 100%.
  • CMV yield after freeze ⁇ drying % CMV concentration after freeze ⁇ drying / mL / CMV concentration before freeze ⁇ drying / mL
  • the monodispersity of a CMV system after the freeze-drying process can be monitored through the GSD ratio value. For instance, a good monodispersity is assessed when the GSD value after freeze-drying is similar to t the one before freeze-drying, resulting in a GSD ratio close to 1. Generally, higher GSD ratios (i.e. closer to 1) indicate a preservation of the initial monodispersity in the CMV distribution after freeze-drying.
  • microvesicles prepared according to the method of the invention may be used in a variety of diagnostic and/or therapeutic techniques, including in particular Ultrasound and Magnetic Resonance.
  • Diagnostic methods include any method where the use of the gas-filled microvesicles allows enhancing the visualisation of a portion or of a part of an animal (including humans) body, including imaging for preclinical and clinical research purposes.
  • imaging techniques may be employed in ultrasound applications, for example including fundamental and harmonic B-mode imaging, pulse or phase inversion imaging and fundamental and harmonic Doppler imaging; if desired three-dimensional imaging techniques may be used.
  • Microvesicles according to the invention may typically be administered in a concentration of from about 0.01 to about 1.0 ⁇ L of gas per kg of patient, depending e.g. on their respective composition, the tissue or organ to be imaged and/or the chosen imaging technique.
  • This general concentration range may of course vary depending on specific imaging applications, e.g. when signals can be observed at very low doses such as in colour Doppler or power pulse inversion.
  • said suspension of gas-filled microvesicles comprises an amphiphilic material and a mixture of freeze-drying protecting component.
  • Reconstitution of the freeze-dried product is preferably made by dispersing it into a physiologically acceptable aqueous carrier, e.g. saline, in the presence of a physiologically acceptable gas, e.g SF 6 , under gentle agitation.
  • a physiologically acceptable aqueous carrier e.g. saline
  • a physiologically acceptable gas e.g SF 6
  • Possible other diagnostic imaging applications include scintigraphy, light imaging, and X-ray imaging, including X-ray phase contrast imaging.
  • Another aspect of the invention relates to the use in a method of therapeutic treatment of a suspension of microvesicles reconstituted from freeze-dried product according to the invention.
  • Therapeutic techniques include any method of treatment (as above defined) of a patient which comprises the combined use of ultrasounds and gas-filled microvesicles either as such (e.g. in ultrasound mediated thrombolysis, high intensity focused ultrasound ablation, blood-brain barrier permeabilization, immunomodulation, neuromudulation, radiosensitization) or in combination with a therapeutic agent (i.e. ultrasound mediated delivery, e.g.
  • a drug or bioactive compound for the delivery of a drug or bioactive compound to a selected site or tissue, such as in tumor treatment, gene therapy, infectious diseases therapy, metabolic diseases therapy, chronic diseases therapy, degenerative diseases therapy, inflammatory diseases therapy, immunologic or autoimmune diseases therapy or in the use as vaccine
  • the presence of the gas-filled microvesicles may provide a therapeutic effect itself or is capable of enhancing the therapeutic effects of the applied ultrasounds, e.g. by exerting or being responsible to exert a biological effect in vitro and/or in vivo, either by itself or upon specific activation by various physical methods (including e.g. ultrasound mediated delivery).
  • Microvesicles according to the invention can typically be administered for therapeutic purposes in a concentration of from about 0.01 to about 5.0 ⁇ L of gas per kg of patient, depending e.g. from their respective composition, the type of subject under treatment, the tissue or organ to be treated and/or the therapeutic method applied.
  • Gas-filled microvesicles were synthesized using a commercially available microfluidic flow-focusing device (CU4553.007 N30 design, Micronit Microfluidics, NL), mounted in a commercially available chip holder (Micronit microfluidics, Fluidic Connect PRO Chip Holder with 4515 Inserts).
  • the microvesicles formation channel had a width of 19 ⁇ m.
  • the chip and its holder were positioned in an optically transparent temperature controlled water bath that was mounted on an inverted microscope equipped with a 20 times magnification objective (Olympus, LMPLAN 20x) and a CCD camera (Lumenera, LM156M). The temperature of the thermostatic bath was set at 50 °C.
  • amphiphilic materials in the liquid flow were: DSPC:DPPE-PEG5000 in a respective molar ratio of 9:1.
  • the materials were added with the above molar ratios at a concentration of 20 mg/mL to a 2:1 (volume ratio) chloroform/methanol mixture under stirring at 60 °C until complete dissolution the amphiphilic material.
  • the solvent was then evaporated under reduced pressure and the obtained film was dried overnight under reduced pressure.
  • the dried material was then redispersed (at concentrations of 15 mg/mL) in saline (0.9% NaCl) at 60 °C under stirring for 30 minutes.
  • the dispersion was then sonicated by using a tip sonicator (Branson Sonifier 250) to homogenously disperse the material.
  • the preparations were then filtered using a polycarbonate filter (0.45 ⁇ m pore size), cooled down to room temperature and degassed.
  • Gas-filled microvesicles comprising CO 2 /C 4 F 10 in a volume ratio of 85/15 were prepared with a gas-mixing device similar to the one schematically illustrated in Figure 3 . Briefly, two gas containers were filled with CO 2 and C 4 F 10 , respectively. The gas flow of each gas was regulated by respective mass flow controllers: (i) EL-Flow: F200CV-002-RAD-11-K, for the CO 2 and (ii) Low- ⁇ P-Flow: F-200DV-RAD-11-Z for C 4 F 10 (both gas controllers from Bronkhorst, Ruurlo, The Netherlands).
  • the mass flow controllers were controlled by a customized software program implemented in Matlab (Mathworks), which was installed on a personal computer, in order to set and keep the desired mixing ratio.
  • a pressure sensor PSE530-M5-L; SMC Corp., Tokyo, Japan
  • the liquid co-flow rate was controlled by using a separate mass flow controller (Mini Cori Flow: M13V14I-MAD-11-K-S; Bronkhorst, Ruurlo, The Netherlands).
  • a liquid co-flow rate of around 150 ⁇ L/min was used to operate the flow-focusing device in the jetting regime and produce microvesicles with a diameter (mode) of around 4 ⁇ m.
  • the calibrated microvesicles suspended in the solution of freeze-drying protecting components were then aliquoted in DIN8R glass vials (1.5 mL suspension/vial) and transferred in the freeze dryer.
  • the vials were cooled at temperatures between -30 °C and -60 °C (as detailed in the subsequent examples) and freeze-dried for approximately 1 hour under vacuum. At the end of the procedure, a freeze-dried composition was obtained as a white homogenous dry solid. The head space was then filled with pure C 4 F 10 .
  • Table 1 lists the freeze-drying protecting components which were investigated. Table 1: Selected freeze-drying protecting components, classified by chemical class Molecule Class Molecular Weight (g/mol) PEG4000 Polymer 4'000 PEG8000 Polymer 8'000 Xylitol Polyol 152.2 Sorbitol Polyol 182.2 Mannitol Polyol 182.2 Glucose Monosaccharide 180.2 Maltose Disaccharide 342.3 Sucrose Disaccharide 342.3 Dextran 6000 Polysaccharide 6'000
  • Freeze-drying protecting components Total concentration of the freeze-drying protecting components (mg/mL) S1A PEG4000 100 S2A PEG8000 100 S3A Sorbitol 100 S4 Xylitol 100 S5 Mannitol 100 M1 (S1A+S3A) PEG4000+Sorbitol 200 M2 (S2A+S3A) PEG8000+Sorbitol 200 M9 (S1A+S4) PEG4000+Xilitol 200 M3 (S2A+S4) PEG8000+Xylitol 200 M4 (S2A+S5) PEG8000+Mannitol 200 S6 Sucrose 100 S7A Maltose 100 S8 Raffinose 100 S9 Dextran 6000 100 M5 (S2A+S6) PEG8000+Sucrose 200 M6 (S2A+S7A) PEG8000+Maltose 200 M7 (S2A+S8) PEG8000+Raffinose 200 M8
  • each freeze-dried composition was reconstituted with 1.5 mL of aqueous solution in the presence of a biocompatible gas, in order to obtain a calibrated and stable microvesicles suspension.
  • concentration of the mixture of freeze-drying protecting components was 188 mg/mL, before and after freeze drying.
  • the reconstituted calibrated microvesicles suspensions were let 5 min on the bench before being characterized using a Coulter Counter Multisizer 3 fitted with a 30 ⁇ m aperture tube, to measure the size, the geometric standard deviation (GSD), the concentration of microvesicles and the yield after the freeze-drying process.
  • GSD geometric standard deviation
  • the GSD value and the concentration of microvesicles were measured before and after the freeze-drying process to evaluate the efficacy of the freeze-drying protecting components in preserving the initial characteristics of the calibrated microvesicles.
  • freeze-drying efficiency was improved using a mixture of freeze-drying protecting components.
  • freeze-drying of calibrated microvesicles in mixtures of polyethylene glycols (PEG4000 and PEG8000) and polyols (i.e. xylitol, sorbitol, and mannitol) enabled to improve the microvesicles yield after freeze-drying.
  • the use of the mixtures M1 allowed an increase of 17% of the microvesicles yield after freeze-drying, compared to the use of S1A.
  • the use of M2 enabled a higher increase of the CMV yield of 38%, compared to the formulation S2A.
  • freeze-drying the CMV in the mixture M2 allowed to obtain a better GSD value of 1.17, lower than those obtained using the single freeze-drying protecting component of the formulations S2A and S3A, 1.21 and 1.33, respectively.
  • the preparation of the freeze-dried composition was furtherly investigated evaluating different freezing temperatures at which the freshly-prepared calibrated microvesicles are cooled at the beginning of the lyophilization procedure.
  • Example 2 The preparation process was performed as previously described in Example 2, except that the vials were cooled at different freezing temperatures, as reported in Table 5.
  • each lyophilized composition was reconstituted with 1.5 mL of aqueous solution in the presence of a biocompatible gas, in order to obtain a calibrated and stable microvesicles suspension.
  • the freeze-dried composition is thus reconstituted with a volume of aqueous solution similar to the volume of suspension which underwent the freeze-drying process. Accordingly, the concentration of the freeze-drying protecting components in the reconstituted suspension is substantially the same as the one in the initial suspension.
  • the reconstituted calibrated microvesicles suspensions were let 5 min on the bench before being characterized using a Coulter Counter Multisizer 3 fitted with a 30 ⁇ m aperture tube, to measure the size, the geometric standard deviation (GSD), the concentration of microvesicles and the yield after the freeze-drying process.
  • GSD geometric standard deviation
  • Table 5 reports the GSD value and the microvesicles yield measured after freeze-drying for each microvesicles suspension obtained after reconstitution of lyophilized compositions frozen at different temperatures and comprising different freeze-drying protecting components.
  • the CMV freeze-dried in the mixture M2 were characterized by the lowest GSD values at any investigated freezing temperature, confirming the results previously obtained in the Example 3.
  • the concentration of the freeze-drying protecting components mixture was also investigated in order to evaluate the freeze-drying efficiency, in terms of preservation of GSD value and microvesicles yield after freeze-drying.
  • freeze-drying protecting components were compared with the corresponding freeze-drying protecting component alone.
  • freeze-drying protecting components were evaluated:
  • the calibrated microvesicles suspended in the different solutions of freeze-drying protecting components were then aliquoted in DIN8R glass vials (1.5 mL suspension/vial) and transferred in the freeze dryer.
  • the vials were cooled at -60°C or -40°C (PEG8000 mixtures) and -60°C (PEG4000 mixtures) for 1 hour at ambient pressure, and followed by primary drying at -20 °C and 0.1 mbar. At the end of the procedure, a freeze-dried composition was obtained as a white homogenous dry solid. The headspace was then filled with pure C 4 F 10 .
  • Equation 2 The CMV yield after freeze-drying (Equation 2) and the GSD ratio (Equation 3) were taken into consideration to study the effect of the concentration of the freeze-drying protecting components on calibrated microvesicles characteristics.
  • Table 6 reports the comparison between the CMV yield after freeze-drying and the GSD ratio for the FD protecting mixture PEG4000 and sorbitol and the PEG4000 alone at increasing concentrations.
  • results showed that an increasing improvement of the freeze-drying efficiency was obtained using concentrations of freeze-drying protecting components between 50 mg/mL and 200 mg/mL for both the mixture and the single components. Moreover, results showed that substantially similar CMV yields are obtained for the mixture PEG4000 and sorbitol and the PEG4000 alone at the investigated freezing temperature ( ⁇ 200 mg/ml).
  • Table 7 reports the comparison between the CMV yield after freeze-drying and the GSD ratio for the FD protecting PEG4000 and xylitol mixture and the PEG4000 alone at increasing concentrations.
  • Table 7 Comparison between PEG4000 alone (S1A) and PEG 4000+xylitol mixture (M9) FD protecting components concentration (mg / mL) Freezing T° CMV Yield after FD GSD ratio PEG4000 PEG4000+Xilitol PEG4000 PEG4000+Xilitol 50 -60°C 18% 44% 0.879 0.900 100 -60°C 42% 53% 0.887 0.926 150 -60°C 69% 55% 0.894 0.926 200 -60°C 65% 66% 0.887 0.932 250 -60°C 65% 61% 0.897 0.922
  • Table 8 reports the comparison between the CMV yield after freeze-drying and the GSD ratio for each investigated concentration by comparing the formulations containing the mixture of PEG8000 and sorbitol and that containing PEG8000 alone.
  • CMV yields were found to be increased only for the mixture at low concentration (50-100 mg/mL) while the values are substantially similar at higher concentration (especially ⁇ 200mg/mL) for both the mixture and the single component.
  • Table 9 reports the comparison between the CMV yield after freeze-drying and the GSD ratio for each investigated concentration by comparing the formulations containing the PEG8000 and xylitol mixture and that containing PEG8000 alone.
  • GSD ratios were found to be improved in the mixture at all concentrations and at both freezing temperatures.

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Claims (12)

  1. Gefriergetrocknete Zusammensetzung umfassend i) ein amphiphiles Material, das ein Phospholipid umfasst, und ii) eine Getriertrocknungs-Schutzkomponente, die bei Rekonstitution mit einer pharmazeutisch annehmbaren Lösung in Gegenwart eines biokompatiblen Gases eine Suspension von kalibrierten gasgefüllten Mikrovesikeln bereitstellt, wobei die Gefriertrocknungs-Schutzkomponente eine Konzentration zwischen 10 und 25 % (Gew./Vol.-%) aufweist und ein Gemisch von wenigstens zwei Gefriertrocknungs-Schutzkomponenten umfassend ein Polyethylenglycol und ein Polyol oder ein Saccharid ist und wobei die rekonstituierte Suspension von kalibrierten gasgefüllten Mikrovesikeln eine geometrische Standardabweichung (GSD) von kleiner als 1,2 aufweist.
  2. Gefriergetrocknete Zusammensetzung gemäß Anspruch 1, wobei das Polyethylenglycol ein Molekulargewicht in dem Bereich zwischen 2000 und 10000 g/mol aufweist.
  3. Gefriergetrocknete Zusammensetzung gemäß Anspruch 2, wobei das Polyethylenglycol ein Molekulargewicht in dem Bereich zwischen 4000 und 8000 g/mol aufweist.
  4. Gefriergetrocknete Zusammensetzung gemäß Anspruch 1, wobei das Polyol Sorbit oder Xylit ist.
  5. Gefriergetrocknete Zusammensetzung gemäß Anspruch 1, wobei das Saccharid Saccharose ist.
  6. Gefriergetrocknete Zusammensetzung gemäß einem der vorstehenden Ansprüche, wobei die rekonstituierte Suspension von kalibrierten Mikrovesikeln durch eine Konzentration von 2,5 × 108 Mikrovesikeln/ml gekennzeichnet ist.
  7. Suspension von gasgefüllten Mikrovesikeln, erhalten durch Rekonstituieren einer gefriergetrockneten Zusammensetzung gemäß einem der vorstehenden Ansprüche mit einer pharmazeutisch annehmbaren Lösung in Gegenwart eines biokompatiblen Gases.
  8. Verfahren zur Herstellung einer gefriergetrockneten Zusammensetzung für die Herstellung einer rekonstituierten Suspension von kalibrierten gasgefüllten Mikrovesikeln mit einer geometrischen Standardabweichung (GSD) von kleiner als 1,2, umfassend die Schritte:
    a. Herstellen einer Suspension von kalibrierten gasgefüllten Mikrovesikeln mit einer geometrischen Standardabweichung (GSD) von kleiner als 1,2 und umfassend ein Gemisch von Gefriertrocknungs-Schutzkomponenten umfassend ein Polyethylenglycol und ein Polyol oder ein Saccharid; und
    b. Gefriertrocknen der Suspension von kalibrierten Mikrovesikeln.
  9. Verfahren gemäß Anspruch 8, wobei das Verfahren zur Herstellung Schritt a. ein mikrofluidisches flussfokussierendes Verfahren umfasst.
  10. Verfahren gemäß Anspruch 8, wobei das Gemisch von Gefriertrocknungs-Schutzkomponenten eine Gesamtkonzentration in dem Bereich zwischen 120 mg/ml und 250 mg/ml aufweist, wobei die Gesamtkonzentration vorzugsweise 200 mg/ml beträgt.
  11. Gefriergetrocknete Zusammensetzung für die Herstellung einer Suspension von kalibrierten gasgefüllten Mikrovesikeln, wobei die gefriergetrocknete Zusammensetzung durch ein Verfahren erhältlich ist, das die folgenden Schritte umfasst:
    a. Herstellen einer ersten Suspension von gasgefüllten kalibrierten Mikrovesikeln mit einer geometrischen Standardabweichung (GSD) von kleiner als 1,2 durch ein fluidfokussierendes Verfahren, wobei die Suspension ferner ein Gemisch von Gefriertrocknungs-Schutzkomponenten umfassend ein Polyethylenglycol und ein Polyol oder ein Saccharid umfasst;
    und
    b. Gefriertrocknen der Suspension.
  12. Verfahren zur Herstellung eines injizierbaren Kontrastmittels, das eine Suspension von gasgefüllten Mikrovesikeln umfasst, wobei das Verfahren Rekonstituieren einer gefriergetrockneten Zusammensetzung gemäß einem der vorstehenden Ansprüche 1-7 mit einer pharmazeutisch annehmbaren Lösung in Gegenwart eines biokompatiblen Gases umfasst.
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